DOI:http://dx.doi.org/10.7314/APJCP.2013.14.2.795 Suppression of Ku80 Correlates with Radiosensitivity and Shortening in U2OS Osteosarcoma Cells

RESEARCH ARTICLE

Suppression of Ku80 Correlates with Radiosensitivity and Telomere Shortening in the U2OS Telomerase-negative Osteosarcoma Cell Line Liu Hu1&, Qin-Qin Wu1&, Wen-Bo Wang1, Huan-Gang Jiang1, Lei Yang1, Yu Liu1, Hai-Jun Yu1, Cong-Hua Xie1,2, Yun-Feng Zhou1,2, Fu-Xiang Zhou1,2*

Abstract

Ku70/80 heterodimer is a central element in the nonhomologous end joining (NHEJ) DNA repair pathway, Ku80 playing a key role in regulating the multiple functions of . It has been found that the Ku80 located at is a major contributor to radiosensitivity in some telomerase positive human cancer cells. However, in ALT human osteosarcoma cells, the precise function in radiosensitivity and telomere maintenance is still unknown. The aim of this study was to investigate the effects of Ku80 depletion in the U2OS ALT cell line cell line. Suppression of Ku80 expression was performed using a vector-based shRNA and stable Ku80 knockdown in cells was verified by Western blotting. U2OS cells treated with shRNA-Ku80 showed lower radiobiological parameters (D0, Dq and SF2) in clonogenic assays. Furthermore, shRNA-Ku80 vector transfected cells displayed shortening of the telomere length and showed less expression of TRF2 protein. These results demonstrated that down-regulation of Ku80 can sensitize ALT cells U2OS to radiation, and this radiosensitization is related to telomere length shortening.

Keywords: Ku80 - telomerase negative osteosarcoma - U2OS cells - radiosensitivity - telomere length - TRF2

Asian Pacific J Cancer Prev, 14 (2), 795-799

Introduction enhanced the radiosensitivity in many cell lines (Yang et al., 2008). However, the cell lines selected in those studies Ku80 is one subunit of the Ku80/ heterodimer. were almost telomerase positive, the effect of Ku80 on The crucial role of Ku80 in DNA damage was well known, the telomere and radiosensitivity in telomerase negative especially the NHEJ pathway. When DNA is damaged, Ku cell lines are still unclear. The aim of this study was to heterodimer recruits DNA-PKcs to the DSB and activates investigate the relationship between radiosensitivity, its kinase function. And then, this process stimulates telomere length and Ku80 protein in telomerase negative DNA repair and the damage signal pathway, which may cancer cells. subsequently affect apoptosis and cell cycles. Besides its important role in DNA repair, many reports have suggested Materials and Methods that Ku80 is also involved in telomere maintenance. When DNA is damaged, the Ku heterodimer protects exposed Cell lines and culture conditions. DNA ends by end-to-end bridging at sites of DNA DSBs. The human osteosarcoma cell U2OS were obtained Unlike the DNA DSBR (double-strand DNA break repair) from the China center for type cultrue collection(CCTCC) pathway, end-to-end joining of DNA should be avoided in Wuhan University. The cells were cultured in RPMI during repair of exposed telomeric ends (Gullo et al., 1640 medium (Sigma chemical Co., St Louis, MO, USA) 2006). During telomere repair, Ku heterodimer bind to at 37℃ supplement with 10% fetal bovine serum and 1% telomeric sequences (Bianchi et al., 1999; Hsu et al., penicillin/streptomycin mixture. All cultures were grown 1999) and promote the telomerase-mediated synthesis of at 37℃ under a humidified atmosphere of 5% carbon the complimentary strand of DNA that prevents end-to- dioxide. end DNA fusion (Bailey et al., 1999; Samper et al., 2000). Previous studies demonstrated that mutation in Ku80 Ku80 shRNA vector constructs proteins prevent repair of telomere ends (Polotnianka et Plasmid pGenesil-1 expression vector (Wuhan genesil al., 1998) and these results reduced telomere lengthen and biotechnology Co, Ltd.) was used, which contained a human

1Department of Radiation and Medical Oncology, Zhongnan Hospital, Wuhan University, 2Hubei Key Laboratory of Tumor Biological Behaviors, Wuhan University, Wuhan, China &Equal contributors *For correspondence: [email protected] Asian Pacific Journal of Cancer Prevention, Vol 14, 2013 795 Liu Hu et al U6 , a GFP reporter and a neomycin resistance whole cell was determined using a BCA protein assay kit gene to enable antibiotic selection in mammalian cells. We (Beyotime biotechnology). The protein extracts (50μg) designed two pairs of complementary oligonucleotide were incubated in loading buffer (60 mmol/L Tris-Hcl, sequences according to the cDNA sequences of Ku80 25% Glycerol, 2% SDS, 14.4 mmol/L Mercaptoethanol, (GenBank accession number: NM_021141). They 0.1% Bromophenol blue), and boiled for 5 min. These analyzed BLAST research to ensure that they didn’t samples were electrophoresed on a 10% SDS-PAGE. have significant with other . After electrophoresis, proteins were transferred to a Target sequences of Ku80 for shRNA are showed below: PVDF membrane and processed for immunoblotting. For 5’-CACCGCCAGGTTCTCAACAGGCTGTTCAAGA the detection of Ku80 and β-actin, blots were incubated CGCAGCCTGTTGAGAACCTGG TTTTTTG-3’ 5’- AG with 1:300 dilution mouse monoclonal antibodies AGCTCAAAAAACCAGGTTCTCAACAGGCTGCGT (Santa cruze biotechnology) and further incubated with CTTGAACAGCCTGTTGAGAACCTGGC-3’. Horseradish peroxidase-conjugated secondary antibody diluted at 1:5000 and specific bands were visualized by shRNA vector stable transfection ECL (Beyotime biotechnology), Autoradiographs were One hundred thousand U2OS cells in a total volume recorded onto X-Omat AR film (Eastman kodak Co.) The of 2 ml RPMI 1640 medium were seeded in 6-well plates density of bands in the resulting film was quantified using and incubated for 24 h. The cells were rinsed with PBS the Image J analysis program. and then were treated with shRNA and scrambled control according to the manufacturer’s instructions. shRNA Real-time quantitative PCR plasmids and Oligofectamine 2000 (Invitrogen, USA) Genomic DNA was extracted from cells using were mixed separately with opti-MEM and incubated for TIANamp genomic DNA kit (TIANGEN biotech Co,. 5 min at room temperature. These reagents were combined Ltd. ) and stored at -70℃. Relative telomere length was and incubated for another 20 min before adding to the detected by using the approach described by Cawthon cells. The effectiveness of transfection was visualized by previously (Cawthon., 2002). This method measures fluorescence microscope 24 h after transfection. After 48 h the factor by which the ratio of telomere repeat copy incubation, 500 μg/ml of hygromycin (G418) was added to number to single-gene copy number differs between the the medium for selection individual colonies which were sample and the reference sample. PCR amplification was isolated after addition 2 weeks culture. The colonies which achieved using telomere (T) and single copy gene(S), contained GFP were selected by fluorescence microscope 36B4 (encodes acidic ribosomal phosphoprotein) primers and continue to culture for at least 10 passages. As a which serves as a quantative control. The mean telomere control, a subset of U2OS cells was transfected with the repeat gene sequence to a reference single copy gene is control plasmid carrying scrambled sequence. represented as T/S ratio which calculated to determine the relative telomere length. Standard curves were generated Semi-quantitative reverse transcription-PCR (RT-PCR) for telomere lengths and the single gene copy amplification Total RNA was isolated using TRIzol reagent reactions from a reference DNA sample serially diluted (Invitrogen) according to the manufacture’s instructions. with Milli-Q water by 2-fold per dilution to produce four The first strand of cDNA was obtained using Revert concentrations of DNA ranging from 50 to 3.125 ng/μl. aidTM first strand cDNA synthesis kit (Fermentas). Duplicate PCR reactions using the TaKaRa realtime PCR For quantitative analysis of Ku80 mRNA, human kit (TaKaRa biotechnology Co, Ltd) according to the GAPDH gene was used as an internal control. DNA manufacture’s instructions. The primers for telomeres primer sequences were designed as follows: F: and the single copy gene 36B4 were added to final 5’-GGGGTACCATGGTGCGGTCGGGGAAT-3’, R: concentrations of 0.2 μM. The telomere and single copy 5’-GCTCTAGATCCCCATACATCCACGAC-3’for gene specific primers used for the experiment were given Ku80, F: 5’-ATCACTGCCACCCAGAAGAC-3’, R: as below: Tel 1: 5’- GGTTTTTGAGGGTGAGGGTGA 5’-AGCGTCAAAGGTGGAGGAGT-3’for GAPDH. GGGTGAGGGTGAGGGT -3’; Tel 2: 5’-TCCCGACTA Amplification cycles were 94℃ for 3 min; the 30 cycles TCCCTA TCCCTATCCCTATCCCTATCCCTA -3’; at 94℃ for 30 sec, 57℃ for 30 s, 72℃ for 1 min, 72℃ for 36B4u: 5’-CAGCAAGTGGGAAGGTGTAATCC-3’, 5 min. PCR products were identified using electrophoresis 36B4d: 5’-CCCATTCTATCATCAACGGGTACAA-3’. on 1.5% agarose gels containing 1% ethidium bromide The cycling conditions consisted of a preincubation for 5 (EB). Gel images were obtained using Bio-ID gel analysis sec at 95℃ and followed by 35 cycles of 15 sec at 95℃, 2 software (Vilber lourmat, France). All experiments were min at 54℃. All experiments were repeated at least three repeated at least three times. times. Thermal amplification was carried out on Mx3000P (Stratagene) and the result was analyzed using the MXP Western blot analysis 3000 analysis program. Cultured cells were rinsed twice with phosphate buffered saline (PBS) and mixed with 200 μl of lysis Grouping and Irradiation buffer (Beyotime biotechnology). The cells in lysis buffer The cells were divided into 3 groups: untreated cells in the dish were removed using a scraper and transferred (negative control), Cells stable transfected with shRNA- to an Eppendorf tube. The cells were homogenized and Ku80, shRNA-scramble. Cells were seeded in 6 wells and centrifuged 1,2000 rpm for 5 min , and the supernatant incubated at 37℃ under humidified 5% CO2, 95% air in were stored at -20℃. The protein concentration of the culture medium. Cells were exposed to γ –rays from a 796 Asian Pacific Journal of Cancer Prevention, Vol 14, 2013 DOI:http://dx.doi.org/10.7314/APJCP.2013.14.2.795 Suppression of Ku80 Correlates with Radiosensitivity and Telomere Shortening in U2OS Osteosarcoma Cells

100.0 Figure 1. Ku80 Gene Down-regulation in U2OS Cells. 6.3 12.8 Figure 2. Clonogenic Assays. Survival curves in U2OS 10.1 20.3 RT-PCR detected Ku80 expression in U2OS cells treated with 10.3 shRNA-Ku80, shRNA-scrambl and negative control. (A) RT- cells treated with shRNA-Ku80, shRNA-scramble and negative PCR was used to detect the Ku80 in the different control. An appropriate number cells in different groups were75.0 25.0 30.0 groups. (B) The PCR products were semiquantified for relative plated into 6-well plates. Each group of cells were irradiated at the dose point of 0, 1, 2, 4, 6, 8, 10 Gy respectively. After 14days 46.8 75.0 levels of mRNA using image analysis by comparing Ku80 with 56.3 51.1 GAPDH. (C) Ku80 protein expression was detected by Western of incubation, the colonies were fixed and stained with crystal 51.7 violet (1% in absolute alcohol). Those colonies containing >5050.0 54.2 blotting in the different groups. (D) The bar chart shows the 31.3 30.0 semiquantitative analysis of Ku80 protein expression. Data cells were scored as viable colonies. The data were fit into the represent means ±SD. P*<0.05 is considered significant single-hit multi-target model, and survival curve of each group were demonstrated by Graphpad prism 5.0 software. 25.0 60Co-ray source (Atomic energy of Canada Ltd, Canada Table 1. Radiobiological Parameters in Different 38.0 31.3 31.3 30.0 33.1 and located in Institute, Seoul, Korea) at a dose rate of Groups 23.7 25.0 27.6 61.3 cGy/min. Grouping D0 Dq SF2 0 0 Colony formation assay shRNA-Ku80 2.106±0.033* 1.028±0.012* 0.602±0.009* An appropriate number of cells which stable shRNA-scarmble 2.384±0.040 2.967±0.015 shRNA-KU80 None Negative control 2.421±0.042 2.692±0.198 0.883±0.007

transfected with shRNA-Ku80, shRNA-scramble and Remission Data from triplicate clonogenic assay were fit into the single- untreated cells were plated into 6-well plates. Each group Radiotherapy of cells were irradiated at the dose point of 0, 1, 2, 4, 6, hit multi-target model, and survival curves of both groups were Chemotherapy 8, 10 Gy respectively. After 14 days of incubation, the drawn by software Graphpad Prism, the SF2, D0, Dq values were calculated (PD0, Dq, SF2<0.05). Data were expressed as colonies were fixed and stained with crystal violet (1% P the mean±SD. *<0.05 is considered significant. SF, surviving recurrence or Persistence in absolute alcohol). Those colonies containing >50 cells fraction; D0, mean lethal dose, is known as final slope; Dq, chemoradiation Concurrent were scored as viable colonies. The data were fit into the quasi-threshold dose single-hit multi-target model, and survival curve of each withtreatment diagnosed Newly group were demonstrated by Graphpad prism 5.0 software. Radiobiological parameters of clonogenic assay. withouttreatment diagnosed Newly Radiobiological parameters, such as D 0, Dq and SF 2were The survival curves describe the radiobiological calculated according to the survival curves. parameters of each group (Figure 2). Compared to the negative control and shRNA-scramble group, the survival Statistic analysis fractions of shRNA-Ku80 group were much lower at All of the experiments were replicated three times. each point. The radiobiological parameters calculated are Data were obtained from triplicate samples and expressed showed in Table 1. D0, Dq and SF 2 values in shRNA- as the mean ±standard deviation. Statistical analysis were Ku80 group are significantly lower than the control groups performed by two-factor ANOVA. Statistical analysis was (P<0.05). Negative control and shRNA-scramble groups performed using software SPSS 13.0 and Graphpad prism showed no significantly changed. The result suggested that 5.0 software. P<0.05 was considered to be statistically downregulating Ku80 protein expression could enhance significant. the radiosensitivity of U2OS cells.

Results Relative telomere length Telomere length was determined from U2OS cell Efficient knockdown of Ku80 in U2OS cells stable transfected shRNA-Ku80 plasmid, shRNA- RT-PCR and western blot were used to analyze the scramble plasmid and negative control by using real-time Ku80 expression. Compared with negative control group PCR. The dissociation curve showing a unique peak from and shRNA-scramble group, the expression of Ku80 the PCR amplification of telomere and single copy gene transcripts (Figure 1A and Figure 1B) and Ku80 proteins (36B4) attested to the specificity of the amplification. The (Figure 1C and Figure 1D) were reduced in shRNA-Ku80 standard curve derived from the amplification of various group. However, negative control cells and shRNA- concentrations indicated that there was linear relationship scramble cells showed no apparent change of Ku80 between Ct values and DNA copy number, suggesting expression. The results indicated that the shRNA-Ku80 that our realtime PCR approach would be useful for could downregulate the expression of Ku80 efficiently in quantifying (data not shown). The relative telomere length gene and protein level. among the cells stable transfected with shRNA-Ku80, Asian Pacific Journal of Cancer Prevention, Vol 14, 2013 797 Liu Hu et al expression. Compared with negative control group and shRNA-scramble group, the expression of TRF2 protein (Figure 4) was reduced in shRNA-Ku80 group. However, negative control cells and shRNA-scramble cells showed no change. The results suggested that downregulating the Ku80 expression could depress the TRF2 expression.

Discussion

Ku80 is a versatile regulatory protein that has been shown to play a crucial role in multiple nuclear processes such as DNA repair, telomere maintenance and apoptosis. Recent reports suggested that there is a positive relationship between Ku80 and telomere length in various cancer cells (Nimura et al., 2007). However, Figure 3. Effect of Suppression of Ku80 on Telomere significant discoveries on the role of Ku80 in telomerase Length in U2OS Cells. Realtime PCR detected the relative negative cancer cells have been uncovered. To study the telomere length in U2OS cells treated with shRNA-Ku80, role of Ku80 in telomerase negative cancer cells, we have shRNA-scrambl and negative control. The Bar graph shows successfully constructed shRNA-Ku80 vector and selected the mean±SD value of relative telomere length. P*<0.05 is the positive colonies cells to suppress the endogenous considered significant Ku80 expression efficiently. In this study, we used the vector-based shRNA expression systems in our research, overcoming the limitations of transient transfection of small interference RNA. Ku80 is an important DNA repair protein in the NHEJ pathway with the other proteins such as DNA–PKcs, XRCC4 and DNA Ligase IV. Ku80 is a critical nuclear protein that not only involved in DNA repair, but also it is essential for the maintenance of telomere length. During telomere repair, Ku80 bind to telomere sequences (Bianchi et al., 1999; Hsu et al., 1999) and associate with human telomerase reverse transcriptase, which is essential for the addition of telomeric repeat sequences (Chai et al., 2002). Moreover, the direct relationship between Ku80 and telomerase has been shown in recent reports (Hsu et al., 2000; Bailey et al., 2004). Suppression of Ku80 expression resulted in losing of telomerase activity, shortening the ends gradually, and apoptosis (Lucero et al., 2003; Kim., 2008). The human telomerase complex is a ribonucleoprotein containing an integral RNA (hTR), a reverse transcriptase protein subunit (hTERT), and other associated proteins. It is reported that Ku protein associated with hTERT physically to protect telomere ends and regulate the access of telomerase to telomere DNA ends (Chai et al., 2002). However, in telomerase negative Figure 4. Effect of Ku80 Down-regulation on The cancer cells, how does the Ku80 protein works without the Expression of TRF2 Protein in U2OS Cells. (A)Western association of hTERT? Here, we have demonstrated that blotting analysis shows the protein expression of TRF2 in U2OS in the telomerase negative cancer cells U2OS, decreased cells treated with shRNA-Ku80, shRNA-scramble and negative Ku80 expression exhibit significant telomere shortening. control. (B) The bar chart shows the semiquantitative analysis Yet, the control group remained unchanged. It means that of Ku80 protein expression. Data represent means±SD. P*<0.05 in the cancer cells lacking of telomerase, Ku80 is probably is considered significant a positive factor in telomere maintenance. It is reported that following targeted Ku80 knockdown, the steady shRNA-scramble and negative control was determined state levels of TRF2 protein which bind to chromatin to be 1.07±0.07, 4.42±1.30, 4.11±0.84, respectively. are reduced. It implicates that Ku80 protein enhances (Figure 3) The result suggested that downregulation of TRF2 chromatin association. Our results also manifested Ku80 protein expression could significantly shorten the that the expression of TRF2 protein were depressed by telomere length. down-regulating the expression of Ku80 protein in U2OS cells. Therefore, it is supposed that Ku80 protein will The protein expression of TRF2 in different groups maintain telomere length with TRF2 protein together. Western blot were used to analyze the TRF2 Nevertheless, the underlying molecular mechanisms of 798 Asian Pacific Journal of Cancer Prevention, Vol 14, 2013 DOI:http://dx.doi.org/10.7314/APJCP.2013.14.2.795 Suppression of Ku80 Correlates with Radiosensitivity and Telomere Shortening in U2OS Osteosarcoma Cells telomere maintenance of Ku80 in telomerase negative Boulton SJ, Jackson SP (1998). Components of the Ku- cancer cells remain unknown and require further research. dependent non-homologous end-joining pathway are In our study, the effect of silencing Ku80 gene on involved in telomeric length maintenance and telomeric radiosensitivity was also investigated. D0 and SF2 values silencing. EMBO J, 17, 1819-28. Cawthon RM (2002). Telomere measurement by quantitative in U2OS cells transfected shRNA-Ku80 plasmid are PCR. Nucleic Acids Res, 30, e47. significantly lower than the control group (P<0.01). This Chai W, Ford LP, Lenertz L, et al (2002). Human Ku70/80 result suggested that Ku80 depletion enhanced the U2OS associates physically with telomerase through interaction cells sensitivity to radiation. Ku80 contributes to genomic with hTERT. J Biol Chem, 277, 47242-7. stability by binding to DNA ends. This binding represents Fink LS, Lerner CA, Torres PF, et al (2010). Ku80 facilitates a key, initiating step in the non-homologous end joining chromatin binding of the telomere binding protein, TRF2. (NHEJ) pathway for DNA repair (Fink et al., 2010). The Cell Cycle, 9, 3798-806. binding results in recruitment of the catalytic subunit Gullo C, Au M, Feng G, et al (2006). The biology of Ku and its of DNA-dependent protein kinase DNA-PKcs to form potential oncogenic role in cancer. Biochim Biophys Acta, 1765, 223-34. the holoenzyme DNA-PK, followed by the exonuclease Hsu HL, Gilley D, Blackburn EH, et al (1999). Ku is associated Artemis and the XRCC4-like factor XLF (Silver et al., with the telomere in . Proc Natl Acad Sci USA, 2012). In addition to its role in DNA repair, multiple lines 96, 12454-8. of evidence indicate that the Ku heterodimer functions Hsu HL, Gilley D, Galande SA, et al (2000). Ku acts in a unique at the telomere length (Boulton et al., 1998). A great way at the mammalian telomere to prevent end joining. number of studies manifested that cellular radiosensitivity Genes Dev, 14, 2807-12. is related to the cellar reproductive ability, so there Kim H (2008). DNA repair Ku proteins in gastric cancer cells should be some kind of intrinsic correlation between and pancreatic acinar cells. Amino Acids, 34, 195-202. the telomere length and radiosensitivity (Barwell et al., Lucero H, Gae D, Taccioli GE (2003). Novel localization of the DNA-PK complex in lipid rafts: a putative role in the signal 2007; Zongaro et al., 2008). Our previous research also transduction pathway of the ionizing radiation response. J revealed that radiosensitivity negatively correlated with Biol Chem, 278, 22136-43. telomere length, the longer telomere length, the lower Nimura Y, Kawata T, Uzawa K, et al (2007). Silencing Ku80 radiosensitivity. (Zhong et al., 2008). It is believed that using small interfering RNA enhanced radiation sensitivity telomere is a key structure to determine the radiosensitivity in vitro and in vivo. Int J Oncol, 30, 1477-84. (Zhou et al., 2010). So, Ku80 depletion may be radiation Polotnianka RM, Li J, Lustig AJ (1998). The yeast Ku sensitizer which would become a radiotherapeutic strategy. heterodimer is essential for protection of the telomere In conclusion, our result indicated that Ku80 depletion against nucleolytic and recombinational activities. Curr enhanced telomerase negative cancer cells U2OS radiation Biol, 8, 831-4. Samper E, Goytisolo FA, Slijepcevic P, et al (2000). Mammalian sensitivity by shortening the telomere length. So Ku80 Ku86 protein prevents telomeric fusions independently of depletion may be radiation sensitizer, which would the length of TTAGGG repeats and the G-strand overhang. become a potential therapeutic strategy. However, the EMBO Rep, 1, 244-52. underlying molecular mechanisms of this action of Ku80 Silver PA, Iglehart JD, Marto JA, et al (2012). DNA ends remain unclear, we believe that this mechanism is worthy alter the molecular composition and localization of Ku of further research multicomponent complexes. Mol Cell Proteomics, 11, 411-21. Yang QS, Gu JL, Du LQ, et al (2008). ShRNA-mediated Ku80 Acknowledgements gene silencing inhibits cell proliferation and sensitizes to gamma-radiation and mitomycin C-induced apoptosis in This study is supported by the National Natural esophageal squamous cell carcinoma lines. J Radiat Res, Science Foundation of China (NO 81172129), the 49, 399-407. Excellent Youth foundation of Hubei Scientific Zhong YH, Liao ZK, Zhou FX, et al (2008). Telomere length Committee(NO 2008CDB126) and PhD. Student inversely correlates with radiosensitivity in human Independent Scientific Research Program of Wuhan carcinoma cells with the same tissue background. Biochem University (NO 20103030201000211). Biophys Res Commun, 367, 84-9. Zhou FX, Xiong J, Luo ZG, et al (2010). cDNA expression analysis of a human radiosensitive-radioresistant cell References line model identifies telomere function as a hallmark of radioresistance. Radiat Res, 174, 550-7. Bailey SM, Brenneman MA, Halbrook J, et al (2004). The Zongaro S, Verri A, Giulotto E, et al (2008). Telomere length kinase activity of DNA-PK is required to protect mammalian and radiosensitivity in human fibroblast clones immortalized telomeres. DNA Repair, 3, 225-33. by ectopic telomerase expression. Oncol Rep, 19, 1605-9. Bailey SM, Meyne J, Chen DJ, et al (1999). DNA double- strand break repair proteins are required to cap the ends of mammalian . Proc Natl Acad Sci USA, 96, 14899-904. Barwell J, Pangon L, Georgiou A, et al (2007). Is telomere length in peripheral blood lymphocytes correlated with cancer susceptibility or radiosensitivity? Br J Cancer, 97, 1696-700. Bianchi A, de Lange T (1999). Ku binds telomeric DNA in vitro. J Biol Chem, 274, 21223-7.

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